The long term objective of the work is to develop improved methods of identifying and characterizing biomolecules, and to apply these techniques to various biological problems. The focus of our effort is on time-of-flight mass spectrometry, (TOFMS), and in applications of mass spectrometry in proteomic and genomic research. TOF instruments have an effectively unlimited mass range and high sensitivity, so they are well suited for the examination of biomolecules. Ions are produced by electrospray ionization (ESI) or matrix-assisted laser desorption/ionisation (MALDI); both techniques can produce ions from biomolecules in the megadalton mass range. Our laboratory has constructed several TOF spectrometers for this purpose, and has been one of the pioneers in the use of "orthogonal injection", where ions are introduced into the TOF instrument perpendicular to the axis. We adopted this technique first for ESI and recently introduced it for MALDI. It has demonstrated significant advantages compared to the usual axial injection method. An important contribution of our laboratory has been the use of collisional cooling of ions in a quadrupole ion guide before injection into the TOF spectrometer; such an arrangement appears to be essential for obtaining optimum results, particularly for MALDI. In addition, the laboratory has coupled a quadrupole mass analyzer (Q) and collision cell (q) to a TOF mass spectrometer to make a QqTOF instrument that is especially useful for studies of biomolecular structure. The application of collisional cooling enables MALDI ions (as well as ESI ions) to be injected into the QqTOF instrument, opening up new possibilities for measuring the structure of MALDI ions. At present this configuration is available only in our laboratory at Manitoba. Proposed applications are concerned with determining the structures of various proteins, including virus coat proteins. We are also developing methods for high throughput identification of gel-separated proteins that cannot be analyzed efficiently by conventional methods. Particular emphasis will also be placed on the study of noncovalent interactions, for which the ESI TOF instrument described above is especially useful. Various protein-ligand, protein-protein and protein-DNA complexes will be examined, and the mass limits of such measurements will be probed.